In modern biotechnology, it is common practice to clone DNA sequences from biological organisms of any type and then to introduce genetic constructs carrying those sequences into plasmids or viral vectors for replication in vitro. Often these sequences are assembled into expression vectors which are then introduced into and expressed in foreign hosts of any of a number of organisms both eukaryotic and prokaryotic. In its isolated form, DNA can be studied, and its sequence can be determined. From a DNA sequence the structure and encoding capacity and other attributes of the DNA can be analyzed. It is also possible to synthesize altered and/or synthetic DNA sequences to make new gene products and to alter the genetic sequence of organisms both large and small.
In the process of expressing a coding sequence of DNA to make a protein, a first step involves the process of transcription whereby a messenger RNA sequence is made, which is ultimately translated into protein. Since the DNA and RNA are essential parts of the protein production process, it is undesirable during the process of in vitro cloning and expression of these nucleotides that the nucleotide chains be degraded. Nevertheless, since imperfectly purified biological reagents are conventionally used in such in vitro processes, the inadvertent introduction of unwanted enzymes is a very practical problem. All organisms make in their cells enzymes known as ribonucleases, which have the principal function of degrading nucleic acids in the cells. Such degradation is an essential part of biological processes both to down regulate messenger RNA which is no longer desired and also as a part of the cellular recycling process in which the component parts of nucleic acids are reused to synthesize other nucleic acids. Therefore, ribonucleases are ubiquitous in biological organisms. Ribonucleases also tend to be stable and highly active. Even trace amounts of ribonucleases can be lethal to in vitro DNA expression systems or systems for handling, utilizing or characterizing RNA, since even a trace amount of a ribonuclease can rapidly degrade all of the mRNA in an experimental sample.
Accordingly, companies which specialize in selling products to researchers in modern biotechnology supply reagents which are specifically intended to help overcome the problem of contaminating trace ribonucleases. Several companies sell, for example, ribonuclease-free water. There is a market for ribonuclease-free water because normal tap water can often be contaminated with extremely small amounts of ribonucleases, which can nevertheless severely disrupt experiments sensitive to such enzymatic activity. Experimenters hands contain ribonucleases which, through insufficiently careful lab techniques, can be introduced to and contaminate the results of carefully done in vitro experiments. Thus the avoidance of contamination by the action of ribonucleases is a significant consideration in many types of experiments in molecular biology.
Ribonucleases can be inhibited by protein molecules produced by cells, the specific purpose of which is to inhibit the enzymatic activity of a ribonuclease. Such proteins are called, naturally enough, ribonuclease inhibitors (or RI). The desirability of ribonuclease inhibitors for use in laboratory techniques of modern biotechnology has led to purified ribonuclease inhibitors being commercial products currently sold on the market by several reagent supply companies. Ribonuclease inhibitor can be isolated from many types of cells, notably most conveniently from placental cells, or it can be created by in vitro expression of DNA sequence which encodes ribonuclease inhibitor. U.S. Pat. No. 5,552,302 describes methods for the production of human recombinant placental ribonuclease inhibitor in prokaryotic cells.
It is a limitation on the ribonuclease inhibitors currently on the market place that they are not very stable, and certainly not as stable as the ribonucleases which they inhibit. Ribonuclease inhibitors tend to be susceptible to rapid oxidation. The oxidation of the ribonuclease inhibitor is a rapid cascading process which is irreversible. The ribonuclease inhibitor has to be completely reduced to bind to a ribonuclease. Since oxygen is, of course, prevalent in the environment, as are many oxidizing agents, this oxidation sensitivity is a severe limitation on the use of ribonuclease inhibitors that reduces their convenient use in laboratory practice of modern techniques of biotechnology. Accordingly, a ribonuclease inhibitor having less susceptibility to oxidation would be more advantageous, because it would be more stable and therefore more likely to decrease the loss of valuable nucleotides to the activity of unwanted ribonucleases.